Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified preparations of a protamine protein kinase from bovine kidney cytosol [Damuni, Amick & Sneed (1989) J. Biol. Chem. 264, 6412-6416] were inactivated after incubation with near-homogeneous preparations of protein phosphatase 2A1 and protein phosphatase 2A2. These protein phosphatase 2A-mediated inactivations of the protamine kinase were unaffected by highly purified preparations of inhibitor 2, but were prevented when the incubations were performed in the presence of 100 nM microcystin-LR, 100 nM okadaic acid or 0.2 mM-ATP. By contrast, highly purified preparations of protein phosphatase 2B, protein phosphatase 2C, the catalytic subunit of protein phosphatase 1, and two forms of a protein tyrosine phosphatase, designated PTPase 1B and T-cell PTPase, had little effect, if any, on protamine kinase activity. Purified preparations of the protamine kinase did not react with anti-phosphotyrosine antibodies, as determined by Western blotting and immunoprecipitation analysis. The results indicate that protein phosphatase 2A is a specific protamine-kinase-inactivating phosphatase.
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PMID:Protein phosphatase 2A is a specific protamine-kinase-inactivating phosphatase. 133 80

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.
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PMID:Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion. 167 49

Protein phosphatases 1 and 2A (PP1 and PP2A) were identified in a variety of plant cells and found to be particulate or soluble depending on the species. In extracts prepared from oilseed-rape seeds these enzymes were associated with microsomes and more rapidly sedimenting fractions, whereas in wheat leaf extracts they were largely microsomal, the remainder being present in the soluble fraction. In pea leaf and carrot cell extracts PP1 and PP2A were almost entirely soluble. No PP1 or PP2A activity was associated with the membranes or stroma of chloroplasts in oilseed-rape seeds, pea leaves and wheat leaves. An Mg2(+)-dependent okadaic acid-insensitive protein phosphatase that resembles protein phosphatase 2C (PP2C) was detected in carrot cells, pea leaves and wheat leaves, but not in oilseed-rape seeds. In wheat leaf extracts PP2C was mostly present in the soluble fraction, a different location from PP1 or PP2A. The rapid inactivation of the cytosolic enzyme quinate dehydrogenase (QDH) in a fraction prepared from light-grown carrot cells was completely blocked by either okadaic acid or microcystin (two potent and specific inhibitors of PP1 and PP2A), whereas inhibitor 2 (a specific inhibitor of PP1) inhibited inactivation by only about 10%. Addition of the purified PP2A catalytic subunit from mammalian skeletal muscle increased the rate of QDH inactivation, whereas addition of mammalian PP1 did not. It is concluded that PP2A is the major enzyme responsible for dephosphorylating (inactivating) QDH in carrot cells. These observations indicate that okadaic acid and microcystin may be useful for identifying other plant processes that are controlled by phosphorylation/dephosphorylation mechanisms. Okadaic acid did not prevent the rapid inactivation of phosphoribulokinase or activation of glucose-6-phosphate dehydrogenase in a fraction prepared from light-grown pea leaves, and addition of the purified catalytic subunits of PP1 and PP2A did not accelerate either process. These observations, in conjunction with the absence of PP1 and PP2A activity in chloroplasts, suggest that these phosphatases are not involved in the regulation of chloroplast metabolism.
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PMID:Plant protein phosphatases. Subcellular distribution, detection of protein phosphatase 2C and identification of protein phosphatase 2A as the major quinate dehydrogenase phosphatase. 184 22

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.
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PMID:Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. 184 81

The synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.
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PMID:An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A. 215 67

Protein phosphatase 2C was isolated from rabbit skeletal muscle by a procedure that involved chromatography on DEAE-cellulose, precipitation with ammonium sulphate, gel-filtration on Sephadex G-100, affinity chromatography on thiophosphorylated myosin-P-light-chain--Sepharose and chromatography on Mono Q. The enzyme was purified about 35,000-fold and 0.3-0.4 mg was isolated from 2500 g skeletal muscle within 5 days. The final step resolved the activity into two peaks, termed protein phosphatases 2C1 and 2C2, that possessed identical substrate specificities and enzymatic properties. About 2.5-fold more protein phosphatase 2C2 was isolated than protein phosphatase 2C1. Protein phosphatases 2C1 and 2C2 migrated as single bands on SDS/polyacrylamide gels yielding apparent molecular masses of 44 kDa and 42 kDa, respectively, and the native proteins were both monomeric at pH 7.5 as judged by their elution from Sephadex G-100 and Sephacryl S200. Peptide maps of protein phosphatases 2C1 and 2C2, obtained after separate digestions with four different proteinases, were different, indicating that they are isoenzymes. Protein phosphatases 2C1 and 2C2 were purified from rabbit liver by the same procedure, and 0.2 mg (2C1 + 2C2) was isolated from 120 g hepatic tissue. Hepatic protein phosphatases 2C1 and 2C2 were also isolated in a molar ratio of about 1:2.5, and their enzymatic properties and apparent molecular masses in the presence and absence of SDS were identical to the skeletal muscle enzymes. Protein phosphatases 2C1 from muscle and liver displayed identical peptide maps, as did protein phosphatases 2C2 from these two tissues. It is concluded that the same two isoenzymes of protein phosphatase 2C are present in skeletal muscle and liver.
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PMID:Identification of two isoenzymes of protein phosphatase 2C in both rabbit skeletal muscle and liver. 303 50

The two recently discovered forms of protein phosphatase 2C, termed 2C1 and 2C2, were digested with CNBr or trypsin, and several peptides corresponding to two regions of the protein were sequenced. These studies revealed close homology between the two enzymes with 49 identities over the 62 residues that could be compared directly. The results establish that protein phosphatases 2C1 and 2C2 are the products of different genes. The C-terminus of protein phosphatase 2C2 has also been identified.
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PMID:Primary structure analysis proves that protein phosphatases 2C1 and 2C2 are isozymes. 304 Jan 25

Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and protamine kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.
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PMID:Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2A from bovine kidney. 753 97

The properties of the calcium/calmodulin-dependent protein phosphatase calcineurin and its potential role in stimulus-secretion coupling were examined in AtT20 mouse pituitary corticotrope tumor cells. Protein phosphatase activity was assayed by measuring the liberation of 32P from 32P-casein, adrenocorticotropin secretion was measured by radioimmunoassay. About 60% of the total phosphatase activity was inhibited by 500 nM okadaic acid, suggesting the presence of protein phosphatases 1 and/or 2A. A further 25-30% reduction of phosphatase activity was achieved by chelating free calcium. Addition of the EF-hand protein blocker trifluoperazine or a calcineurin autoinhibitory peptide fragment markedly reduced okadaic acid resistant and calcium-dependent protein phosphatase activity indicating that calcium-dependent 32P release is largely due to calcineurin (protein phosphatase 2B). The remaining 10-15% of total activity was Mg2+ dependent and blocked by NaF, hence possibly due to protein phosphatase 2C. Calcineurin activity was inhibited by the immunosuppressants FK506 and cyclosporin A, either when added to the cell lysates or after preincubation of intact cells with the drugs for 30 min at 37 degrees C. When added to lysates, cyclosporin A inhibited calcium/calmodulin-dependent phosphatase more effectively than FK506. However, when tested on intact cells, FK506 proved 10-fold more potent than cyclosporin A. Both immunosuppressive agents enhanced the calcium-dependent release of adrenocorticotropic hormone into the medium, once more, FK506 was 10-fold more potent than cyclosporin A. Taken together, these data suggest that calcineurin is an inhibitory element in the signal transduction pathway controlling exocytotic secretion in pituitary cells that express voltage-operated calcium channels. This is in direct contrast with leukocytes where voltage-operated calcium channels are not found, and calcineurin is an important element for agonist-induced activation.
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PMID:Inhibitory role for calcineurin in stimulus-secretion coupling revealed by FK506 and cyclosporin A in pituitary corticotrope tumor cells. 768 29

The abnormally phosphorylated forms of tau factor are major constituents of neurofibrillary tangles in Alzheimer's disease brain. In order to investigate protein phosphatases which are related to dephosphorylation of abnormal phosphorylation sites, we examined the dephosphorylation of tau factor phosphorylated by three proline-directed type protein kinases. Tau factor phosphorylated by cdc2 kinase and tau protein kinase II was dephosphorylated by the holoenzyme of protein phosphatase 2A and calcineurin, while either the catalytic subunit of protein phosphatase 2A or protein phosphatase 2C could not catalyze the dephosphorylation. From the kinetic analysis, we concluded that tau factors phosphorylated by the protein kinases serve as good substrates for protein phosphatase 2A and calcineurin. On the other hand, tau factor phosphorylated by glycogen synthase kinase 3 alpha was dephosphorylated by the catalytic subunit of protein phosphatases 2A as well as the holoenzyme of protein phosphatase 2A and calcineurin. It has been reported that serines 199, 202 and 396 according to the numbering of the longest human tau isoform are among the major abnormal phosphorylation sites of tau factor. We synthesized two phosphopeptides which contained phosphoserines 199 and 202 or phosphoserine 396 and prepared the polyclonal antibodies specific for the phosphopeptides. Using these antibodies, we confirmed that the holoenzyme of protein phosphatase 2A and calcineurin could dephosphorylate phosphoserines 199, 202 and 396 in tau factor. The catalytic subunit of protein phosphatase 2A could dephosphorylate phosphoserine 396 but not phosphoserines 199 and 202. Neurofibrillary tangles in Alzheimer's disease brain were immunostained with both antibodies but the normal neurons in the normal aged brains were not. The results suggest that protein phosphatase 2A and calcineurin can be involved in the dephosphorylation of abnormal phosphorylation sites in tau factor and that the dephosphorylation of phosphoserine 396 is differently regulated from phosphoserines 199 and 202.
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PMID:Dephosphorylation of abnormal sites of tau factor by protein phosphatases and its implication for Alzheimer's disease. 778 67


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